An important class of microscopy image contrast techniques is based on the phenomenon of photoluminescence. It well known that when certain molecules are exposed to an exciter light (typically a high power, short wave ultra-violet or blue light), they will emit a longer wavelength light (i.e., fluorescent light). Fluorescence (and/or phosphorescence) may occur naturally in a sample, for example, chlorophyll in botanical specimens, or may be induced through the use of particular binding agents such as dyes or other such fluorochromes. In conventional fluorescence microscopy, an excitation filter is typically employed to generate the exciter light from a polychromatic illumination source, and a sharp barrier or emission filter is typically employed to permit only the fluorescent light to reach the ocular or camera (i.e., the viewer). Thus, for example, a short-wave blue light exciter filter in conjunction with a red barrier filter enables chlorophyll-containing organelles to appear brilliant red over a relatively dark background.
One of the shortcomings with conventional fluorescence microscopy, however, is that the information which is provided to the viewer only indicates the area or region where fluorescent light is emitted from the sample. The conventional technique does not inform the viewer as to where the light is absorbed, which may be different from where light is emitted. This difference may arise, for example, due to some type of tunneling mechanism or other unexplained phenomenon.
Accordingly, it would be desirable to be able to image a sample in order to ascertain where the exciter light is being absorbed, and to ascertain if fluorescent emitting structure is or is not co-located with the fluorescent absorbing structure. This would enable the microscopist to determine if the sample does indeed exhibit some type of tunneling effect.